DNA vectors, including bacterial plasmids, are found naturally along with genomic DNA in prokaryotes and sometimes in eukaryotic organisms. Plasmids are double-stranded, circular DNA molecules that replicate within a cell independently of chromosomal or genomic DNA of the cell. Plasmid size can vary from 1 K base pairs to over 1,000 K base pairs. Plasmids contained within a cell are identical. However, the numbers of plasmid copies within a particular cell can range anywhere from a single unit to several thousand copies.
One way to classify plasmids is by function. Fertility-F-plasmids, which contain tra-genes, are capable of conjugation (transfer of genetic material between bacteria which are touching). Resistance-(R)-plasmids contain genes that can build a resistance against antibiotics or poisons and help bacteria produce pili. Col-plasmids contain genes that code for bacteriocins, proteins that can kill other bacteria. Degradative plasmids enable the digestion of unusual substances, e.g., toluene or salicylic acid. Virulence plasmids turn the bacterium into a disease causing pathogen.
Plasmid DNA can appear in different conformations. These include “Nicked Open-Circular” DNA with one strand cut. “Relaxed Circular” DNA is fully intact with both strands uncut, but has been enzymatically “relaxed” (supercoils removed). “Linear” DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo. “Supercoiled” (or “Covalently Closed-Circular”) DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form. “Supercoiled Denatured” DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.
Plasmids serve as important tools in genetics and biotechnology labs where they are used in genetic engineering to duplicate DNA sequences or generate new sequences. Plasmids are commonly used to multiply (make many copies of) or express particular genes. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest, which can be induced to produce large amounts of proteins from the inserted gene.
Plasmids in this form are called vectors and are considered transferable genetic elements of cells. They are capable of autonomous replication and expression if placed within a suitable host. Plasmid host-to-host transfer requires direct, mechanical transfer allowing the intentional uptake of the genetic element by transformation into the host. Restriction enzymes are frequently used to specifically cut the plasmid DNA at specific short sequences in open plasmids and insert short sequences of DNA. The inserts can have a number of functions including coding for the production of a particular and desired protein. When researchers manipulate DNA vectors in this manner, it is sometimes called a DNA construct since the researcher has generated a customized DNA sequence.
DNA vectors have been discovered in nature and used the research laboratory as a molecular biology tool. DNA vectors have been classified according to host organism and the origin of the extrachromosomal DNA molecules. When these vectors are used to harbor DNA sequences of interest, these vectors are called cloning vectors. Plasmids are autonomously replicating DNA molecules from bacteria. Plasmids can be engineered to maintain and replicate DNA sequences of interest. These sequences are inserted into the plasmid and the size of the insert is restricted by the type of cloning vector. Plasmids can contain inserts sizes of 1 to 200 kb. Viruses that attack bacteria, bacteriophage, can insert its chromosome into the host bacteria. The bacteria's cellular machinery is then instructed to replicate many copies of the bacteriophage. Researchers have taken advantage of this biology to generate a cloning vector from bacteriophage, which can be used to carry DNA insert sizes up to 16 kb. Taking advantage of the Lambda phage replication system, a plasmid based upon Lambda's replication machinery has been generated called a cosmid. Cosmids can carry up to 25 kp of DNA. Large vectors can be generated from the bacteria fertility plasmid. When these fertility plasmids are generated as engineered DNA constructs, they are called Bacteria Artificial Chromosomes (BACs) and can maintain insert sizes of 150-350 kb. A similar manipulation can be achieved in yeast in which an artificial yeast chromosome is generated. These are called Yeast Artificial Chromosomes (YACs) and can contain DNA inserts between 100 kb and 3,000 kb.
In addition to cloning vectors, many DNA vectors have been used as a method for DNA delivery. When DNA vectors are introduced in bacterial cells, non-animal eukaryotes and plant cells, this is called transformation. Using non-viral methods, such as chemical or electroporation to introduce DNA vectors into eukaryotic cells is called transfection. There are viral methods to introduce DNA vectors into animal cells, which are called transduction. With the ability to use many systems to propagate a DNA vector and transfer it to different organisms, it is important to be able to recover the DNA vectors from all of these different systems. DNA vectors can be isolated from cell lysate of prokaryotes, eukaryote and archea. DNA vectors can also be isolated from cell culture systems such as mammalian and insect cell systems.
In order to manipulate and use DNA vectors for molecular cloning, they often need to be isolated and purified from a cell or cell culture. Current technology tools used to isolate and purify plasmids include the use of solid phase extraction plates, spin columns and magnetic beads. Due to the complexity of the purification process, procedures using these tools consist of a combination of several manual and automated steps.
To start, DNA vectors or plasmids are grown in cell cultures over several hours under controlled heat and nutrient growth conditions. After cell culture growth is complete, the first step in the process is to recover the cells into a pellet. The cells are spun down and cleaned, removing the growth medium and any extra cell culture debris and material. At this point, the cell pellet is re-suspended in a buffer and then is mixed with a lysing solution to break up the cell walls and release the plasmids. An RNase is usually included to digest any RNA that may be present so that it is not captured and purified with the plasmid in later steps. After lysing, the mixture is neutralized. The resulting mixture consists of plasmid, genomic DNA, proteins, cell debris and other miscellaneous materials. The lysing and neutralization process must be performed gently in order to prevent or limit shearing of the genomic DNA. Genomic DNA is large and in effect chemically different from DNA vectors, but sheared genomic DNA may mimic plasmid in subsequent steps and could be captured and contaminate the final recovered DNA vector product. Once the lysing and neutralization steps are completed, the mixture is usually clarified with another spin down step to remove cell debris and genomic material.
At this point, columns, plates or magnetic beads containing a solid extraction phase are used in a series of automated or manual steps to recover the DNA vector from the clarified lysate. Complete automation of the process is not performed mainly because of the need to remove the cellular debris from the sample. There exists a need to reduce the manual manipulation of DNA vector purification.
Solid phase extraction is a powerful technology for purifying and concentrating biomolecules including DNA vectors and including plasmids. Sample preparation methods are needed to permit the automated purification and concentration of DNA vectors. This is difficult because of the presence of cellular debris in the samples. Not only can particulate plug columns but extraneous sample material can interfere with the selective capture of the DNA vector or be co-captured. Spin column methods and magnetic beads process samples from clarified cell lysates where the cell debris is removed from the sample with centrifugation prior to sample processing. The subject invention involves methods and devices for extracting and recovering DNA vectors from a sample using a bed of extraction media contained in a pipette tip column. These methods, and the related devices and reagents, will be of particular interest to the life scientist, since they provide a powerful technology for purifying, concentrating, analyzing and using plasmids and other biomolecules of interest from biological samples including cell culture and in particular un-clarified cell lysates. The methods, devices and reagents can find wide application in a variety of preparative, genetic engineering and analytical contexts.